Detection of survivin in the biological fluids of cancer patients

ABSTRACT

The present invention includes a method for diagnosing cancer comprising detecting the presence of survivin in the biological fluid of a patient. The present invention also provides kits comprising one or more agents that detect survivin polypeptide or survivin nucleic acid and a container for collecting biological fluid for testing.

RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S.Provisional Application 60/260,898, filed Jan. 12, 2001, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of diagnosing cancercomprising detecting the presence of survivin in the biological fluidsof a patient. Specifically, the method relates to the diagnosis ofbladder cancer, comprising detecting the presence of survivin in urinesamples of a patient. Additionally, the present invention relates tokits for diagnosing cancer comprising an agent that detects survivin inthe biological fluids of a patient and a container for collecting thebiological fluids.

BACKGROUND

[0003] Cancer refers to malignant neoplasms that tend to invadesurrounding tissues and that metastasize to new body sites. They arelikely to recur after attempted removal and to cause death of thepatient unless adequately treated. The exact cause of cancer is notknown, but links between certain activities such as smoking or exposureto carcinogens and the incidence of certain types of cancers and tumorshave been shown by a number of researchers.

[0004] The spread of cancer from a primary site to distant organs, i.e.,metastasis, still remains the main cause of death for most cancerpatients. Despite years of research, the genetic mechanisms involved inthe process remain largely uncharacterized. Such information is ofspecial importance in cancer prognosis given the uncertain course of thedisease. Since cancer prognosis cannot always be accurately assessedusing current tumor grading techniques, one of the greatest obstacles tothe successful treatment of the cancer patient continues to be the lackof sound prognostic markers.

Bladder Cancer

[0005] Bladder cancer is a malignant tumor growth which usually arisesfrom the urothelial cells, (transitional cells) that line the bladder.Urothelial carcinoma of the bladder, i.e. bladder cancer, is the fourthmost common cancer in men and the eighth most common cancer in women inthe United States, accounting for more than 54,000 new cases and 11,200deaths every year. Recurrences of bladder cancer occur in up to 80% ofpatients and constitute a formidable obstacle to long-lastingremissions, with such recurrences frequently involving muscle invasionand disseminated disease (Dawson et al., ABC of Urology: UrologicalMalignancies-II: Urothelial Tumors. BMJ, 1996, 312:1090-94).

[0006] Numerous factors may contribute to the development of bladdercancer. Cigarette smoking and occupational exposure to a certain classof organic chemicals called aromatic amines (beta-naphthylamines,xenylamine, 4-nitrobiphenyl, benzidine) are well-established riskfactors. Some studies indicate that there may be a link between highdoses of the artificial sweetener saccharin and transitional cellbladder cancer. Other studies have shown that women who receivedradiation therapy for the treatment of cervical cancer have an increasedrisk of developing transitional cell bladder cancer. Additionally,people who received the chemotherapy drug, cyclophosphamide (Cytoxan),have demonstrated a greater risk of developing bladder cancer.

[0007] The most common clinical presentation of bladder cancer ishematuria. Frequently, however, the diagnosis of bladder cancer isdelayed because the hematuria is either intermittent or attributed toother causes, such as a urinary tract infection or the use ofanti-coagulants. Voided urine cytology of transitional cells isconventionally used to diagnose bladder cancer. If the urinary cytologyis positive, then transitional cell cancer of the urothelium is almostcertainly present. However, cytological examination of transitionalcells may be negative in up to half of the patients with bladder cancer.Thus, negative cytological results do not rule out the presence ofbladder cancer (Badalament et al., Cancer, 1987, 59(12): 2078; Cohen etal., Urologic Clinics of North America, 1992, 19(3):421).

[0008] As an added diagnostic complication, the entire urinary tractmust be evaluated for transitional cell cancer once an initial diagnosisof bladder cancer is made because transitional cells line the urinarytract starting at the level of the kidneys, including the renal pelvis,the ureters, the bladder, and most of the urethra. The renal pelvis ofthe kidneys and ureters is best evaluated by intravenous pyelogram (IVP)or retrograde pyelogram. An IVP involves an intravenous injection ofcontrast material which is then filtered out of the blood into the urineby the kidney. Plain x-rays taken during this process show the urinarytract. Typically a retrograde pyelogram is reserved for patients with anintravenous contrast allergy or poor visualization on IVP. A retrogradepyelogram is performed at the time of cystoscopy.

[0009] Cystoscopy, which involves the use of a lighted instrument toview inside the bladder, is an uncomfortable procedure utilized for theunambiguous diagnosis of bladder cancer. In performing a retrogradepyelogram through the cystoscope a small plastic tube is inserted intothe ureter, and contrast material is injected into the ureter andkidney. Cystoscopy enables the identification of small subtleabnormalities that may be missed by other diagnostic modalities such asultrasound, computed tomography, or magnetic resonance imaging. Thus,office cystoscopy is an essential part of the initial evaluation and atthis time cannot be substituted by other tests. Today, most officecystoscopic examinations are performed with a flexible scope. Comparedto rigid cystoscopy, flexible endoscopy is more comfortable and allowsthe physician to see around the curves of an enlarged prostate.

[0010] A biopsy may also be performed to diagnose bladder cancer. Abiopsy is the removal of a small sample of living tissue from an organ,such as the bladder, for microscopic examination to confirm or establisha diagnosis, estimate prognosis, or follow the course of a disease.However, biopsies are invasive procedures, and are therefore notdesirable and it frequently is necessary for a person to undergoanesthesia. In addition, as with any invasive procedure, an individualundergoing biopsy runs the risk of infection. Further, the entirebladder cannot be biopsied to determine whether bladder cancer ispresent.

Staging Bladder Cancer

[0011] Bladder cancers are classified based on their aggressiveness andhow different they are from the surrounding bladder tissue(differentiation). They are staged and graded by physicians whilediagnosing bladder cancer.

[0012] There are several different ways to stage tumors. The two mostcommonly used staging systems for bladder cancer are the ABCD system(the Jewett-Strong-Marshall system) and the TNM system. The ABCD systemis older and uses A-B-C-D staging to classify the distinct phases orperiods of bladder cancer. The system basically uses the followingscale: 0, carcinoma in situ (tumor limited to the bladder mucosa(lining)); A, tumor extends through the mucosa but does not extendbeyond the submucosa; B, tumor invades the muscle; C, tumor invades intothe fat; and D, cancer has spread to regional lymph nodes or to distantsites. Each letter is followed by a number, for example A1, B2, etc.With the TNM system, the bladder is described by the T, the lymph nodesby the N, and distant spread by the M. Each letter is followed by adescribing number, T2aN0M0. For example, Ta denotes a non-invasivepapillary carcinoma; Tis denotes a carcinoma in situ; and T1 denotes atumor invading subepithelial connective tissue.

[0013] Bladder cancers spread to the rest of the body by extending intothe nearby organs, including the prostate, uterus, vagina, ureters, andrectum. Metastasis occurs through the pelvic lymph nodes, where thetumor next spreads to the liver, lungs and bones.

Grading Bladder Cancer

[0014] Bladder cancers are graded by a pathologist from the biopsy. Thegrade of a cancer provides information regarding how fast the cancermight be growing or how aggressive it might be. High grade cancers growfaster and spread earlier than low grade cancers. The current system ofgrading uses only three different grades: well-differentiated,moderately differentiated, and poorly differentiated (or Grade I, II orIII). Some pathologists will use a 4-level grading system, I, II, IIIand IV. Either system is acceptable, and the pathologist will alwaysnote how many levels they use by declaring the cancer as a II/III orII/IV. The denominator or second number states what system they use. Awell-differentiated tumor means that the cancer has more resemblance tonormal bladder tissue and therefore usually does not grow or spreadquickly. A poorly differentiated tumor means that the cancer does notresemble normal bladder and usually grows quickly and spreads to othertissues earlier. Moderately differentiated tumors are in the middle.

[0015] Grade, while important, has less bearing on the treatmentdecisions than does the stage. After the grade and stage are known,other factors also come into play before making any decision aboutfuture treatment.

Prostate Cancer

[0016] Prostate cancer is a malignant tumor growth within the prostategland. It is the third most common cause of death from cancer among menof all ages and is the most common cause of death from cancer in menover 75 years old. Prostate cancer is rarely found in men younger than40 years of age.

[0017] Although the cause is unknown, some studies have shown arelationship between prostate cancer and high dietary fat intake andincreased testosterone levels. There is no known association with benignprostatic hyperplasia (BPH).

[0018] Like bladder cancer, prostate cancers are classified or stagedbased on their aggressiveness and the degree that they are differentfrom the surrounding prostate tissue. Most prostate cancers are stagedusing the A-B-C-D staging system or the TNM system. For prostate cancer,the A-B-C-D system basically categorizes tumors using the followingscale: A tumor not palpable (able to be felt) but detectable inmicroscopic biopsy; B palpable tumor confined to prostate; C extensionof tumor beyond prostate with no distant metastasis; and D cancer hasspread to regional lymph nodes. The TNM system, on the other hand,describes the prostate (T), the lymph nodes (N), and evidence ofmetastatic disease (distant spread) (M) separately. Prostate cancersspread by extending into the seminal vesicles, bladder, and peritonealcavity. Prostate cancers typically metastasize to the lymph nodes,bones, lungs, liver, and kidneys.

[0019] Today, prostate cancer is usually graded using the Gleasongrading system, named after a pathologist from the University ofMinnesota. The system involves looking for different patterns ofaggressiveness within the prostate and then giving two scores of 1-5.These two scores are added up to give the total Gleason score which willrange from 2-10. The higher the score, the more aggressive the tumorwill be. For example, a typical Gleason graded cancer might be writtenas Gleason 4+3=7, or Gleason 2+2=4.

Survivin

[0020] Deregulated expression of inhibitors of apoptosis (programmedcell death) is thought to contribute to cancer by abnormally extendingcell viability, favoring the accumulation of mutations, and promotingresistance to therapy (Reed, J. Clin. Oncol., 1999, 17:2941-53). A novelmodulator of the cell death/viability balance in cancer was recentlyidentified as survivin (Ambrosini et al., Nat. Med., 1997, 3:917-21), amember of the Inhibitor of Apoptosis (IAP) gene family (Deveraux et al.,Genes Dev., 1999, 13:239-52).

[0021] Survivin is a 16.5 kDa cytoplasmic protein containing a singlepartially conserved BIR (baculovirus IAP repeats) domain, and a highlycharged carboxyl-terminal coiled-coil region instead of a RING finger,which inhibits apoptosis induced by growth factor (IL-3) withdrawal whentransferred in B cell precursors (Ambrosini et al., Nat Med 1997,3:917-921). Based on overall sequence conservation, the absence of acarboxyl-terminal RING finger and the presence of a single, partiallyconserved BIR domain, survivin is the most distantly related member ofthe IAP family, sharing the highest degree of similarity with NAIP(neuronal apoptosis inhibitory protein; Roy et al., Cell, 1995,80:167-178). Additionally, unlike other IAP proteins, survivin isundetectable in normal adult tissues, but becomes the top fourthtranscript expressed in common human cancers (Ambrosini et al., Nat.Med., 1997, 3:917-21; Velculescu et al., Nat. Genet., 1999, 23:387-88),such as lung, colon, breast, pancreas, and prostate, and in ˜50% ofhigh-grade non-Hodgkin's lymphomas, in vivo. Consistent with a proposedrole of deregulated apoptosis in urothelial cancer (Gazzaniga et al.,Int. J. Cancer, 1996, 69:100-04; Lara et al., Int. J. Radiat. Oncol.Biol. Phys., 1999, 43:1015-19), survivin was found in 78% of bladdercancers, but not in normal urothelium, and its expression correlatedwith accelerated recurrences (Swana et al., N. Engl. J. Med., 1999,341:452-53). Because of its over-expression in cancer but not in normaltissues (Ambrosini et al., Nat. Med., 1997, 3:917-21; Velculescu et al.,Nat. Genet., 1999, 23:387-88), and its unfavorable predictive/prognosticsignificance in various malignancies (Adida et al, Lancet, 1998,351:882-83; Islam et al., Oncogene, 2000,19:617-23; Tanaka et al., Clin.Cancer Res., 2000, 6:127-34; Monzo et al., J. Clin. Oncol., 1999,17:2100-04; Kawasaki et al., Cancer Res., 1998, 58: 5071-74), survivinmay constitute a useful molecular marker in cancer. This is particularlyrelevant in bladder cancer (Stein et al., J. Urol., 1998, 160:645-59;Ozen, Curr. Opin. Oncol., 1998, 10:273-78) where simple and non-invasivediagnostic means to monitor response to therapy and simplify follow upprotocols are urgently needed. Although regarded as “gold standard”(Brown, Urol. Clin. North Am., 2000, 27:25-37), urine cytology has a lowsensitivity (30-40%) in bladder cancer, and fails to detect superficial,low-grade lesions. Accordingly, the inventors of the instant applicationinvestigated the potential suitability of bodily fluid survivin as a newmolecular marker for detection of cancer, specifically detection ofurine survivin for the diagnosis of bladder cancer.

SUMMARY OF THE INVENTION

[0022] The present invention provides a method of diagnosing cancer in apatient comprising assaying a sample of biological fluid from a patientfor the presence or absence of survivin, wherein the presence ofsurvivin in the sample indicates that the patient has cancer. In apreferred embodiment, the biological fluid is selected from the groupconsisting of prostatic fluid, seminal fluid, whole blood, serum, urine,breast biopsy fluid, gastrointestinal fluid, and vaginal fluid, and thecancer is new onset or recurrent cancer selected from the groupconsisting of lung cancer, colon cancer, breast cancer, pancreaticcancer, prostate cancer, bladder cancer, renal cancer, genitourinarytract cancer, non-hodgkin's lymphoma, and neuroblastoma. Morepreferably, the biological fluid is urine or blood serum, and the canceris bladder or prostate cancer. Most preferably, the biological fluid isurine, and the cancer is bladder cancer.

[0023] In one aspect, the present invention contemplates a method todetermine the grade of the cancer by quantitating the amount of survivinin the biological fluid of a patient, wherein a high level of survivinindicates that the cancer is of a high grade. Additionally, the presentinvention contemplates the use of the method to determine the stage ofthe cancer, wherein a cancer of the CIS stage has a higher level ofsurvivin than the cancer of the non-invasive papillary carcinoma stage.

[0024] In another aspect, the present invention contemplates a method ofmonitoring cancer in a patient comprising quantitating the amount ofsurvivin in the biological fluid of a patient. Additionally, the presentinvention contemplates determining the prognosis of a patient bydetecting or quantitating the amount of survivin in the biological fluidof the patient. In some instances, the presence of survivin indicates apoor prognosis. Quantitation of survivin includes measuring relativelevels of survivin in a sample. It may also include detection of thepresence or absence of survivin.

[0025] Preferably, the survivin is detected using an agent that binds tosurvivin or hybridizes to the nucleic acid encoding survivin. Mostpreferably, the agent is selected from the group consisting ofantibodies that bind survivin, survivin binding fragments, and nucleicacids that hybridize to a nucleic acid encoding survivin, and the agentis tagged with a label. The label may be a radioactive label, afluorescent label, an enzyme, a chemiluminescent tag, or a colorimetrictag.

[0026] In one aspect of the invention, survivin is detected by animmunoassay. Preferably, the immunoassay is an enzyme linkedimmunosorbent assay or radioimmunoassay, and the immunoassay comprisesimmunoblotting, immunodiffusion, immunoelectrophoresis, orimmunoprecipitation. More preferably, survivin is detected by dotblotting, most preferably, by using the Bio-Dot method and the Bio-DotSF module.

[0027] In another aspect of the invention, survivin is detected bynucleic acid hybridization.

[0028] In a preferred embodiment, the nucleic acid hybridization isRT-PCR or Northern blot analysis. The agent used in nucleic acidhybridization is labeled with a radioactive label, a fluorescent label,an enzyme, a chemiluminescent tag, or a colorimetric tag.

[0029] The present invention also provides kits for diagnosis,prognosis, or monitoring cancer in a patient, comprising a container forcollecting biological fluid from a patient and one or more agents thatdetect the presence of survivin in the biological fluid. The agents inthe kit can be tagged with a label. Alternatively, other components canbe included in the kit for tagging the agents. The present inventionalso contemplates kits comprising other components for diagnosing andmonitoring cancer in a patient, and determining the prognosis of thecancer. In one embodiment, the components of the kit are packaged eitherin aqueous medium or in a lyophilized form.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 shows urine detection of survivin using the Bio-Dot SFmodule. Increasing concentrations of recombinant survivin in μg/ml (leftcolumn), or urine specimens from the indicated patient groups wereapplied to a slot-blot apparatus. The membrane was incubated with anantibody to survivin followed by HRP-conjugated goat anti-rabbit IgG.Bands were visualized by chemiluminescence and quantitated bydensitometry. TCC, bladder cancer (Group 4); TCC/R, remission (Group 5);TCC/T, under treatment (Group 5); RCC, renal cell carcinoma, PC,prostate cancer (Group 3); PSA, patient with rising PSA withoutdiagnosis of prostate cancer (Group 2); BPH, benign prostate hyperplasia(Group 2); Ctrl, healthy volunteers (Group 1).

[0031]FIG. 2 shows Western blotting of urine survivin. Urine cellpellets from a normal healthy volunteer (Normal) and a Group 4 patientwith bladder cancer (TCC) were electrophoresed, transferred to nylonmembranes and immunoblotted with an antibody to survivin followed bychemiluminescence. Relative molecular weight markers are indicated onthe left.

[0032]FIG. 3 shows RT-PCR amplification of survivin mRNA in urine. TotalRNA was extracted from urine cell pellets and reverse-transcribed byrandom priming. Amplification reactions were carried out withsurvivin-specific nested primers (279 bp) or β-actin-specific primers(309 bp). Molecular weight markers in bp are indicated (M). TCC,analysis of 5 representative patients with new or recurrent bladdercancer (Group 4).

[0033]FIG. 4 shows serum detection of survivin using the Bio-Dot SFmodule. Increasing concentrations of recombinant survivin in μg/ml (leftand right column), urine or serum samples were applied to a slot-blotapparatus. The membrane was incubated with an antibody to survivinfollowed by HRP-conjugated goat anti rabbit IgG. Bands were visualizedby chemiluminescence. A number followed by “serum B1 CA” indicates serumfrom patients with TCC. Most of these samples were applied to the blotin duplicate. ⅞ serum samples from patients with bladder cancer werepositive. One sample (8-serum B1 CA) was negative for one of the twosamples tested. Urine and serum from patient 2 B1 CA were positive.“Urine Hx TCC” indicates urine from a single patient previouslydiagnosed with TCC. This patient was resected and is undergoing BCGtreatment. “3-serum P CA” indicates survivin positive serum from apatient with prostate cancer. “6-serum BPH” is serum from a patient withbenign prostate hypertrophy. This sample was survivin positive ½ timesthat it was tested. No addition information is available for thispatient. “Urine TCC” is urine from a patient with bladder cancer. “Negcon” is a protein control. “Blank” is TBS buffer only.

DETAILED DESCRIPTION OF THE INVENTION I. General Description

[0034] The present invention is based on the need to develop safe,reliable non-invasive screening strategies for detecting cancer,specifically bladder cancer. The identification of a singlepredictive/prognostic marker of the disease has remained elusive (Steinet al., J. Urol. 1998, 160: 645-59). The identification of a marker andthe development of a method to diagnose and screen for cancer,especially transitional cell carcinoma of the bladder, as well as formonitoring cancer activity and determining the prognosis of anindividual having bladder cancer would be useful to the treatment ofcancer.

[0035] The present invention discloses a simple, antibody or other probebased test to identify the apoptosis inhibitor, survivin, in the urineor other bodily fluid of patients with cancer.

[0036] The present invention is based in part on the discovery thaturine survivin is found in all patients with newly diagnosed orrecurrent bladder cancer (46/46), but not in normal volunteers (0/17),or in patients with other urologic cancers (0/30), and only in 4/30patients with non-neoplastic genito-urinary disorders. The presentinvention is also based on the finding that of the three patients withhematuria who tested positive for urine survivin, one had a positivecytology for bladder cancer and another one was diagnosed with bladdercancer within six months of survivin detection. Moreover, the presentinvention is based on the finding that patients treated for bladdercancer and achieving cystoscopic remission are negative for urinesurvivin (32/35).

[0037] The present invention is also based in part on the finding thatsurvivin is detected in the blood serum of patients with bladder andprostate cancer.

II. Specific Embodiments 1. Biological Fluids

[0038] As used herein, the term “biological fluids of patients” refersto fluids from living organisms. The term encompasses “bodily fluids”which are found in the body of living organisms.

[0039] The present invention includes the use of agents in the screeningof biological fluids for the presence of survivin. In vitro serologicalevaluation of biological fluids withdrawn from patients thereby permitsnon-invasive diagnosis of cancers. For example, human bodily fluids suchas prostatic fluid, seminal fluid, whole blood, serum, urine, breastbiopsy fluid, gastrointestinal fluid, and vaginal fluid can be takenfrom a patient and assayed for the presence of survivin using an agentthat can detect survivin by radioimmunoassays or enzyme-linkedimmunoassays, competitive binding enzyme-linked immunoassays, dot blot,Western blot, Northern blot, PCR, or other assays known in the art.

[0040] In a preferred embodiment, blood serum from patients is collectedto detect the presence of survivin for diagnosis of bladder or prostatecancer.

[0041] More preferably, urine samples from patients are collected todetect the presence of survivin for diagnosis of bladder cancer. Urinesamples to be tested may be collected well in advance of testing andfrozen prior to treatment. If the urine sample is to be frozen prior toanalysis, it is preferred that the sample be collected and placed onice. Preferably within a time period of about 2 hours, the iced sampleis centrifuged to pellet cellular debris, then filtered, and thefiltrate then promptly frozen at −20° C., more preferably at −80° C.Just prior to analysis, frozen samples should be rapidly thawed in a 37°C. water bath. Fresh urine samples may also be used immediately aftercollection, optimal centrifugation, and filtration. More preferably,whole urine is kept on ice until it is frozen at −80° C. After thawingat 4° C., the urine is centrifuged at 4° C. and assayed for survivin.

2. Agents that Detect Survivin

[0042] Agents that are capable of detecting survivin in the biologicalfluids of patients are those that interact with survivin or the nucleicacid encoding survivin. Examples of such agents include, but are notlimited to survivin antibodies or fragments thereof that bind survivin,survivin binding partners such as p34^(cdc2)-cyclin B1 kinase, andnucleic acids that hybridize to the nucleic acid encoding survivin.

Survivin Antibodies

[0043] Survivin antibodies have been prepared and used by the skilledartisan. Lu et al. (Cancer Res., 1998, 58(9):1808-12) teach thepreparation of mouse monoclonal survivin antibodies from recombinantlyproduced survivin/glutathione S-transferase fusion protein forimmunohistochemical analysis of gastric carcinomas. Grossman et al. (J.Invest. Dermatol., 1999, 113:1076-81) disclose the use of rabbitpolyclonal antibodies to detect survivin in human metastatic malignantmelanoma cell lines.

[0044] Survivin or survivin peptides may be used to raise antibodiesusing standard immunological procedures well known and described in theart (Practical Immunology, Butt, ed., Marchel Dekker, New York, 1984).Briefly, an isolated survivin or survivin peptide produced, for example,by recombinant DNA expression in a host cell, is used to raiseantibodies in a xenogenic host. Preferred antibodies are antibodies thatbind specifically to an epitope on the survivin protein, preferablyhaving a binding affinity greater than about 10⁵ M⁻¹, most preferablyhaving an affinity greater than about 10⁷ M⁻¹ for that epitope. Forexample, where antibodies to a human survivin protein are desired, asuitable antibody generating host is a mouse, goat, rabbit, guinea pig,or other mammal useful for generating antibodies. The survivin proteinor peptide is combined with a suitable adjuvant capable of enhancingantibody production in the host, and injected into the host, forexample, by intraperitoneal administration. Any adjuvant suitable forstimulating the host's immune response may be used. A currentlypreferred adjuvant is Freund's complete adjuvant (an emulsion comprisingkilled and dried microbial cells, e.g., from Calbiochem Corp., SanDiego, or Gibco, Grand Island, N.Y.). Where multiple antigen injectionsare desired, the subsequent injections comprise the antigen incombination with an incomplete adjuvant (e.g. cell-free emulsion).

[0045] In a preferred embodiment, the disclosed method of detecting thepresence of survivin in biological fluids is performed using antibodiesthat bind specifically to survivin. Polyclonal and monoclonal antibodiesthat bind specifically to survivin may be prepared by methods known inthe art. Antibodies include recombinant polyclonal or monoclonal Fabfragments prepared in accordance with the method of Huse et al.(Science, 1989, 246:1275-1281; see also Campbell, “Monoclonal AntibodyTechnology, The Production and Characterization of Rodent and HumanHybridomas” in Burdon et al., Eds, Laboratory Techniques in Biochemistryand Molecular Biology, 1985 Volume 13, Elsevier Science Publishers,Amsterdam).

[0046] As mentioned above, methods for preparing polyclonal andmonoclonal antibodies are well known to the skilled artisan. Briefly,polyclonal antibodies may be produced by injecting a host mammal, suchas a rabbit, mouse, rat, or goat, with the survivin protein or asurvivin peptide or fragment. Sera from the mammal are extracted andscreened to obtain polyclonal antibodies that are specific to thepeptide or peptide fragment.

[0047] The survivin protein, peptide, or fragment for generation ofantibodies may be obtained by isolation from its natural source, byrecombinant means, or by synthetic means.

[0048] In order to produce monoclonal antibodies, a host mammal isinoculated with a survivin protein or peptide and then boosted. Spleensare collected from inoculated mammals a few days after the final boost.Cell suspensions from the spleens are fused with a tumor cell inaccordance with the general method described by Kohler and Milstein(Nature, 1975, 256:495-497). In order to be useful, a peptide fragmentmust contain sufficient amino acid residues to define the epitope of thesurvivin molecule being detected.

[0049] If the fragment is too short to be immunogenic, it may beconjugated to a carrier molecule. Some suitable carrier moleculesinclude keyhole limpet hemocyanin and bovine serum albumin. Conjugationmay be carried out by methods known in the art. One such method is tocombine a cysteine residue of the fragment with a cysteine residue onthe carrier molecule. The peptide fragments may be synthesized bymethods known in the art. Some suitable methods are described by Stuartand Young in “Solid Phase Peptide Synthesis,” Second Edition, PierceChemical Company (1984).

[0050] Purification of the antibodies or fragments can be accomplishedby a variety of methods known to those of skill including, precipitationby ammonium sulfate or sodium sulfate followed by dialysis againstsaline, ion exchange chromatography, affinity or immunoaffinitychromatography as well as gel filtration, zone electrophoresis, etc.(Goding in, Monoclonal Antibodies: Principles and Practice, 2d ed., pp.104-126, Orlando, Fla., Academic Press). It is preferable to usepurified antibodies or purified fragments of the antibodies having atleast a portion of a survivin binding region, including such as Fv,F(ab′)₂, Fab fragments (Harlow and Lane, 1988, Antibody Cold SpringHarbor) for the detection of survivin in the fluids of cancer patients,preferably in the urine of bladder cancer patients.

[0051] For use in detection and/or monitoring of cancer, the purifiedantibodies can be covalently attached, either directly or via linker, toa compound which serves as a reporter group to permit detection of thepresence of survivin. A variety of different types of substances canserve as the reporter group, including but not limited to enzymes, dyes,radioactive metal and non-metal isotopes, fluorogenic compounds,fluorescent compounds, etc. Methods for preparation of antibodyconjugates of the antibodies (or fragments thereof) of the inventionuseful for detection, monitoring are described in U.S. Pat. Nos.4,671,958; 4,741,900 and 4,867,973.

[0052] In one aspect of the invention, preferred binding epitopes may beidentified from a known survivin gene sequence and its encoded aminoacid sequence and used to generate survivin antibodies with high bindingaffinity. Also, identification of binding epitopes on survivin can beused in the design and construction of preferred antibodies. Forexample, a DNA encoding a preferred epitope on survivin may berecombinantly expressed and used to select an antibody which bindsselectively to that epitope. The selected antibodies then are exposed tothe sample under conditions sufficient to allow specific binding of theantibody to the specific binding epitope on survivin and the amount ofcomplex formed then detected. Specific antibody methodologies are wellunderstood and described in the literature. A more detailed descriptionof their preparation can be found, for example, in Practical Immunology,Butt, W. R., ed., Marcel Dekker, New York, 1984.

[0053] The present invention also contemplates the detection of survivinantibodies. Survivin is a cancer specific marker. Thus, detection ofsurvivin antibodies in biological fluids of a patient may enable thediagnosis of cancer.

Survivin Binding Partners

[0054] Other molecules that bind survivin can also be used to detect thepresence of survivin in biological fluids. Examples of survivin bindingpartners, other than survivin antibodies, include but are not limited top34^(cdc2)-cyclin B1 kinase and caspase-9.

Nucleic Acids

[0055] Nucleic acids including naturally occurring nucleic acids,oligonucleotides, antisense oligonucleotides, and syntheticoligonucleotides that hybridize to the nucleic acid encoding survivin,are useful as agents to detect the presence of survivin in thebiological fluids of cancer patients, preferably in the urine of bladdercancer patients. Ambrosini et al. (Nat Med, 1997, 3: 917-921) disclosecloning of the survivin gene. Nucleic acids and oligonucleotides thatare useful agents for the present invention include but are not limitedto those corresponding to the survivin gene isolated by Ambrosini et al.(Nat Med, 1997, 3:917-921). The present invention contemplates the useof nucleic acid sequences corresponding to the coding sequence ofsurvivin and to the complementary sequence thereof, as well as sequencescomplementary to the survivin transcript sequences occurring furtherupstream or downstream from the coding sequence (e.g., sequencescontained in, or extending into, the 5′ and 3′ untranslated regions) foruse as agents for detecting the expression of survivin in biologicalfluids of cancer patients, preferably in the urine of bladder cancerpatients.

[0056] The preferred oligonucleotides for detecting the presence ofsurvivin in biological fluids are those that are complementary to atleast part of the cDNA sequence encoding survivin. These complementarysequences are also known in the art as “antisense” sequences. Theseoligonucleotides may be oligoribonucleotides oroligodeoxyribonucleotides. In addition, oligonucleotides may be naturaloligomers composed of the biologically significant nucleotides, i.e., A(adenine), dA (deoxyadenine), G (guanine), dG (deoxyguanine), C(cytosine), dC (deoxycytosine), T (thymine) and U (uracil), or modifiedoligonucleotide species, substituting, for example, a methyl group or asulfur atom for a phosphate oxygen in the inter-nucleotidephosohodiester linkage. Additionally, these nucleotides themselves,and/or the ribose moieties may be modified.

[0057] The oligonucleotides may be synthesized chemically, using any ofthe known chemical oligonucleotide synthesis methods well described inthe art. For example, the oligonucleotides are prepared by using any ofthe commercially available, automated nucleic acid synthesizers.Alternatively, the oligonucleotides may be created by standardrecombinant DNA techniques, for example, inducing transcription of thenoncoding strand. The DNA sequence encoding survivin may be inverted ina recombinant DNA system, e.g., inserted in reverse orientationdownstream of a suitable promoter, such that the noncoding strand now istranscribed.

[0058] Although any length oligonucleotide may be utilized to hybridizeto a nucleic acid encoding survivin, oligonucleotides typically withinthe range of 8-100 nucleotides are preferred. Most preferableoligonucleotides for use in detecting survivin in urine samples arethose within the range of 15-50 nucleotides.

[0059] The oligonucleotide selected for hybridizing to the survivinnucleic acid, whether synthesized chemically or by recombinant DNAtechnology, is then isolated and purified using standard techniques andthen preferably labeled (e.g., with ³⁵S or ³²P) using standard labelingprotocols.

[0060] The present invention also contemplates the use ofoligonucleotide pairs in polymerize chain reactions (PCR) to detect theexpression of survivin in biological fluids. The oligonucleotide pairsconsist of a survivin primer and a reverse survivin primer. Thepreferred oligonucleotide pairs are SEQ ID NO: 1 and SEQ ID NO: 2. Morepreferably, the oligonucleotide pairs are SEQ ID NO: 3 and SEQ ID NO: 4.

3. Methods of Detection Protein Binding Assays

[0061] As will be appreciated by those skilled in the art, any means forspecifically identifying and quantifying a survivin protein in thebiological fluid of a cancer patient is contemplated. The currentlypreferred means for detecting survivin protein in a sample is by meansof a binding protein capable of interacting specifically with a markerprotein. Preferably, labeled antibodies, binding portions thereof, orother survivin binding partners may be used. The antibodies may bemonoclonal or polyclonal in origin, or may be biosynthetically produced.The survivin binding partners may also be naturally occurring moleculesor synthetically produced. The amount of complexed survivin protein,e.g., the amount of survivin protein associated with the bindingprotein, is determined using standard protein detection methodologiesdescribed in the art. A detailed review of immunological assay design,theory and protocols can be found in numerous texts in the art,including Practical Immunology, Butt, W. R., ed., Marcel Dekker, NewYork, 1984.

[0062] A variety of assays are available for detecting proteins withlabeled antibodies. In a one-step assay, the survivin molecule, if it ispresent, is immobilized and incubated with a labeled antibody. Thelabeled antibody binds to the immobilized target molecule. After washingto remove unbound molecules, the sample is assayed for the presence ofthe label.

[0063] In a two-step assay, immobilized survivin molecule is incubatedwith an unlabeled antibody. The survivin-unlabeled antibody complex, ifpresent, is then bound to a second, labeled antibody that is specificfor the unlabeled antibody. The sample is washed and assayed for thepresence of the label.

[0064] The choice of marker used to label the antibodies will varydepending upon the application. However, the choice of the marker isreadily determinable to one skilled in the art. These labeled antibodiesmay be used in immunoassays as well as in histological applications todetect the presence of tumors. The labeled antibodies may be polyclonalor monoclonal. In a preferred embodiment, the antibodies are polyclonalrabbit antibodies.

[0065] The antibodies may be labeled with a radioactive atom, an enzyme,a chromophoric or fluorescent moiety, or a colorimetric tag. The choiceof tagging label also will depend on the detection limitations desired.Enzyme assays (ELISAs) typically allow detection of a colored productformed by interaction of the enzyme-tagged complex with an enzymesubstrate. Some examples of radioactive atoms include ³²P, ¹²⁵I, ³H, and¹⁴P. Some examples of enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase.Some examples of chromophoric moieties include fluorescein andrhodamine. The antibodies may be conjugated to these labels by methodsknown in the art. For example, enzymes and chromophoric molecules may beconjugated to the antibodies by means of coupling agents, such asdialdehydes, carbodiimides, dimaleimides, and the like. Alternatively,conjugation may occur through a ligand-receptor pair. Some suitableligand-receptor pairs include, for example, biotin-avidin or-streptavidin, and antibody-antigen.

[0066] The most sensitive label known to date is a chemiluminescent tagin which interaction of the tag with a reactant results in theproduction of light. Useful labels include chemiluminescent moleculessuch as acridium esters or chemiluminescent enzymes where the reactantis an enzyme substrate. When, for example, acridium esters are reactedwith an alkaline peroxide solution, an intense flash of light isemitted, allowing the limit of detection to be increased 100 to 10,000times over those provided by other labels. In addition, the reaction israpid. A detailed review of chemiluminescence and immunoassays can befound in Weeks et al. (Methods in Enzymology, 1983, 133:366-387),Kawaguichi et al. (Stabilized Phenyl Acridinium Esters ForChemiluminescent Immunoassay—Bioluminescence and Chemiluminescence,Proceedings of 9th International Symposium 1996, Edited by Hastings,Kricka and Stanley, John Wiley & Sons, 1997, pp. 480-484), and U.S. Pat.No. 5,468,646. Other considerations for fluid assays include the use ofmicrotiter wells or column immunoassays. Column assays may beparticularly advantageous where rapidly reacting labels, such aschemiluminescent labels, are used. The tagged complex can be eluted to apost-column detector which also contains the reactant or enzymesubstrate, allowing the subsequent product formed to be detectedimmediately.

[0067] In one aspect, the present invention contemplates the use of asandwich technique for detecting survivin proteins in serum and otherbiological fluids. As described in PCT Publication WO93/09437, publishedMay 13, 1993, the technique requires two antibodies capable of bindingthe protein of interest: e.g., one immobilized onto a solid support, andone free in solution, but labeled with some easily detectable chemicalcompound. Examples of chemical labels that may be used for the secondantibody include but are not limited to radioisotopes, fluorescentcompounds, and enzymes or other molecules which generate colored orelectrochemically active products when exposed to a reactant or enzymesubstrate. When samples containing the survivin protein are placed inthis system, the survivin protein binds to both the immobilized antibodyand the labeled antibody. The result is a “sandwich” immune complex onthe support's surface. The complexed protein is detected by washing awaynonbound sample components and excess labeled antibody, and measuringthe amount of labeled antibody complexed to protein on the support'ssurface. The sandwich immunoassay is highly specific and very sensitive,provided that labels with good limits of detection are used.

[0068] The present invention also contemplates screening numeroussamples of biological fluids at the same time. This can be performedusing the conventional 96-well microtiter format which is widely usedand easily automatable. There are also several commercially availablespectrometers (“plate readers”) for calorimetrically analyzing 96-wellplates.

[0069] Preferably, the presence of survivin in a sample of bodily fluidis detected by radioimmunoassays or enzyme-linked immunoassays,competitive binding enzyme-linked immunoassays, dot blot, Western blot,chromatography, preferably high performance liquid chromatography(HPLC), or other assays known in the art.

[0070] Dot blotting is routinely practiced by the skilled artisan todetect a desired protein using an antibody as a probe (Promega Protocolsand Applications Guide, Second Edition, 1991, Page 263, PromegaCorporation). Samples are applied to a membrane using a dot blotapparatus. A labeled probe is incubated with the membrane, and thepresence of the protein is detected.

[0071] Western blot analysis is well known to the skilled artisan(Sambrook et al., Molecular Cloning, A Laboratory Manual,1989, Vol. 3,Chapter 18, Cold Spring Harbor Laboratory). In Western blot, the sampleis separated by SDS-PAGE. The gel is transferred to a membrane. Themembrane is incubated with labeled antibody for detection of the desiredprotein.

[0072] The assays described above involve steps such as but not limitedto, immunoblotting, immunodiffusion, immunoelectrophoresis, orimmunoprecipitation.

[0073] Preferably, the survivin is detected by dot blotting or byWestern blotting. More preferably, survivin is detected in thebiological fluid of cancer patients by dot blotting using the Bio-Dotmethod and the Bio-Dot SF module.

Nucleic Acid Detection

[0074] The presence of survivin in a sample of biological fluid of apatient may also be determined by nucleic acid hybridization, such asbut not limited to Northern blot analysis, dot blotting, Southern blotanalysis, fluorescence in situ hybridization (FISH), and PCR.Chromatography, preferably HPLC, and other known assays may also be usedto determine messenger RNA levels of survivin in a sample.

[0075] The survivin DNA conceivably may be found in the biologicalfluids inside a survivin-positive cancer cell that is being shed orreleased in the fluid under investigation.

[0076] In one aspect, the present invention contemplates the use ofnucleic acids as agents for detecting survivin in biological fluids ofpatients, wherein the nucleic acids are labeled. The nucleic agents maybe labeled with a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a colorimetric tag or other labels or tags thatare discussed above or that are known in the art.

[0077] In another aspect, the present invention contemplates the use ofNorthern blot analysis to detect the presence of survivin mRNA in asample of bodily fluid. The first step of the analysis involvesseparating a sample containing survivin nucleic acid by gelelectrophoresis. The dispersed nucleic acids are then transferred to anitrocellulose filter or another filter. Subsequently, the labeledoligonucleotide is exposed to the filter under suitable hybridizingconditions, e.g. 50% formamide, 5×SSPE, 2×Denhardt's solution, 0.1% SDSat 42° C., as described in Molecular Cloning: A Laboratory Manual,Maniatis et al. (1982, CSH Laboratory). Other useful procedures known inthe art include solution hybridization, dot and slot RNA hybridization,and probe based microarrays. Measuring the radioactivity of hybridizedfragments, using standard procedures known in the art quantitates theamount of survivin nucleic acid present in the biological fluid of apatient.

[0078] Dot blotting involves applying samples containing the nucleicacid of interest to a membrane. The nucleic acid can be denatured beforeor after application to the membrane. The membrane is incubated with alabeled probe. Dot blot procedures are well known to the skilled artisanand are described more fully in U.S. Pat. Nos. 4,582,789 and 4,617,261,the disclosures of which are incorporated herein by reference.

[0079] Polymerase chain reaction (PCR) is a process for amplifying oneor more specific nucleic acid sequences present in a nucleic acid sampleusing primers and agents for polymerization and then detecting theamplified sequence. The extension product of one primer when hybridizedto the other becomes a template for the production of the desiredspecific nucleic acid sequence, and vice versa, and the process isrepeated as often as is necessary to produce the desired amount of thesequence. The skilled artisan to detect the presence of desired sequence(U.S. Pat. No. 4,683,195) routinely uses polymerase chain reaction.

[0080] A specific example of PCR that is routinely performed by theskilled artisan to detect desired sequences is reverse transcript PCR(RT-PCR; Saiki et al., Science, 1985, 230:1350; Scharf et al., Science,1986, 233:1076). RT-PCR involves isolating total RNA from biologicalfluid, denaturing the RNA in the presence of primers that recognize thedesired nucleic acid sequence, using the primers to generate a cDNA copyof the RNA by reverse transcription, amplifying the cDNA by PCR usingspecific primers, and detecting the amplified cDNA by electrophoresis orother methods known to the skilled artisan.

[0081] In a preferred embodiment, the methods of detecting survivinnucleic acid in biological fluids of cancer patients, preferably urineof bladder cancer patients, include Northern blot analysis, dotblotting, Southern blot analysis, FISH, and PCR. More preferably, themethod of detection is RT-PCR using the following two sets of primers:SEQ ID NO: 1 and 2 and SEQ ID NO: 3 and 4.

4. Kits for Diagnosing Cancer

[0082] In one aspect, the present invention includes kits comprising therequired elements for diagnosing cancer. Preferably, the kits comprise acontainer for collecting biological fluid from a patient and an agentfor detecting the presence of survivin or its encoding nucleic acid inthe fluid. The components of the kits can be packaged either in aqueousmedium or in lyophilized form.

[0083] Kits for diagnosing or monitoring cancer containing one or moreagents that detect the survivin protein, such as but not limited tosurvivin antibodies, fragments thereof, or survivin binding partners canbe prepared. The agent(s) can be packaged with a container forcollecting the biological fluid from a patient. When the antibodies orbinding partner are used in the kits in the form of conjugates in whicha label is attached, such as a radioactive metal ion or a moiety, thecomponents of such conjugates can be supplied either in fully conjugatedform, in the form of intermediates or as separate moieties to beconjugated by the user of the kit.

[0084] Kits containing one or more agents that detect survivin nucleicacid, such as but not limited to the full length survivin nucleic acid,survivin oligonucleotides, and pairs of survivin primers can also beprepared. The agent(s) can be packaged with a container for collectingbiological fluid from a patient. The nucleic acid can be in the labeledform or to be labeled form.

[0085] Other components of the kit may include but are not limited to,means for collecting biological fluid, means for labeling the agent,membranes for immobilizing the survivin or survivin nucleic acid in thebiological fluid, means for applying the biological fluid to themembrane, means for binding the agent to survivin in the biologicalfluid of a patient, a second antibody, a means for isolating total RNAfrom a biological fluid of a patient, means for performing gelelectrophoresis, means for generating cDNA from isolated total RNA,means for performing hybridization assays, and means for performing PCR,etc.

5. Uses for Assays and Kits of the Present Invention

[0086] The assays or methods and kits of the present invention areuseful for diagnosis, prognosis, and monitoring cancer in a patient. Inone aspect of the invention, the presence of survivin in the biologicalfluid of a patient indicates that the patient has cancer. The cancercould be a new onset cancer or a recurrent cancer. As used herein, theterm “recurrent cancer” means that the cancer has come back (recurred)after it has been treated. As used herein the term “new onset cancer”refers to newly developed cancer.

[0087] Survivin may be detected in biological fluids from any of thecancers where survivin is expressed and secreted, released, or found ina bodily fluid. Survivin is expressed in cancers of the lung, colon,pancreas, breast, prostate (Ambrosini et al., Nat. Med., 1997,3:917-21), bladder (Swana et al., N. Engl. J. Med., 1999, 341:452-53),and in non-Hodgkin's lymphoma. Expression of survivin in neuroblastoma,breast, lung bladder and colorectal cancers correlated with unfavorabledisease and abbreviated survival (Adida et al., Lancet, 1998,351:882-83; Islam et al., Oncogene, 2000, 19:617-23; Tanaka et al.,Clin. Cancer Res., 2000, 6:127-34; Monzo et al., J. Clin. Oncol., 1999,17:2100-04; Kawasaki et al., Cancer Res., 1998, 58:5071-74; Swana et al,N. Engl. J. Med., 1999, 341:452-53).

[0088] Survivin may be released by exfoliated cancer cells into theextracellular environment during tumor progression. Accordingly, anybiological fluid from a patient can be assayed for the presence ofsurvivin. Preferably, human bodily fluids such as prostatic fluid,seminal fluid, whole blood, serum, urine, breast biopsy fluid,gastrointestinal fluid, and vaginal fluid can be taken from a patientand screened for the presence of survivin.

[0089] In a preferred embodiment, the methods and kits of the presentinvention are used to diagnose genitourinary tract cancer includingbladder cancer, prostate cancer, and renal cancer by detecting survivinin the biological fluids of patients. More preferably, the methods andkits of the present invention are used to diagnose bladder cancer bydetecting the presence of survivin in the urine samples of patients. Thepresence of survivin in the urine samples of a patient indicates thatthe patient has bladder cancer.

[0090] In another aspect, the methods and kits of the present inventionmay be used to quantitate the amount of survivin in the biological fluidof a patient. The amount of survivin in the biological fluid of apatient may be useful for grading the cancer. A high level of survivinin the biological fluid of a patient may indicate that the cancer is ofa high grade. Alternatively, the methods and kits of the presentinvention may be used to determine the stage of the cancer in a patient.A cancer of the CIS stage has a higher level of survivin than the cancerof the non-invasive papillary carcinoma stage.

[0091] As used herein, the term “papillary carcinoma” refers to amalignant neoplasm characterized by the formation of numerous,irregular, fingerlike projections of fibrous stroma that is covered witha surface layer of neoplastic epithelial cells.

[0092] As used herein, the term “carcinoma in situ (CIS)” is synonymouswith intraepithelial carcinoma. The term when used to refer to bladdercancer means a flat tumor in the bladder lining (the transitionalcells).

[0093] Further, the amount of survivin quantitated from the biologicalfluid of a patient may be used to monitor the progression of the cancerin the patient and to determine the prognosis of a cancer patient. Forexample, the amount of survivin measured over a period of time providesinformation as to how fast the cancer is growing.

[0094] Urine survivin can be used as a quick and inexpensive method formonitoring patients. The urine survivin test can be integrated in abattery of urine markers to improve the sensitivity and specificity ofearly detection of recurrences.

[0095] It is contemplated that after patients are tested positive forsurvivin in their biological fluids, they will be further tested usingmore invasive and expensive approaches, such as cystoscopy.

[0096] In light of the foregoing general discussion, the specificexamples presented below are illustrative only and are not intended tolimit the scope of the invention. Other generic and specificconfigurations will be apparent to those persons skilled in the art.

EXAMPLES Materials and Methods

[0097] Urine Specimens: One hundred fifty eight urine specimens werecollected at the urology clinics at Yale-New Haven Hospital and at theVeterans Administration, New England Health Care Systems, West Haven,Conn. Division. Random clean-catch or straight catheter urine sampleswere obtained from individuals who were categorized into 5 differentgroups. Group 1, normal healthy volunteers of mean age of 47.6±20.8years taking no medications (n=17). Group 2, patients of mean age of60.0±18.1 years with diagnosis of non-neoplastic urinary tract diseaseor hematuria (n=30). Group 3, patients of mean age of 71.5±9.9 yearswith diagnosis of genitourinary cancer, excluding bladder cancer (n=30).Group 4, patients of mean age of 69.7±8.7 years with diagnosis of newonset or recurrent bladder cancer (n=46). Group 5, patients of mean ageof 76.1±8.9 years who were undergoing treatment or had already receivedtreatment for bladder cancer and had a negative cytoscopic evaluation onthe day of urine collection (n=35). Treatment measures in group 5included intravesical bacillus Calmette-Guerin (BCG), thiotepa,transurethral resection, partial cystectomy and radiation. Group 4included patients who after urine collection underwent similar treatmentmeasures and/or salvage cystectomy or radical cystectomy.

[0098] Statistical Analysis: The relationship between urine survivin andpatients' diagnosis was analyzed by a Chi square test. Non-parametricstatistical analysis was used to compare the weighted urine survivinscore with the grading classification system performed at the Yale-NewHaven Hospital.

Example 1 Urine Detection of Survivin Using the Bio-Dot SF Module

[0099] Urine specimens were filtered onto a nitrocellulose membraneusing a microfiltration apparatus in a module providing a 48-well slotformat. The blot was analyzed for the presence of survivin using apolyclonal antibody. The protocol is as follows: urine was collected andstored at −80° C. until analysis. On the day of analysis, urine sampleswere centrifuged at 20,000×g for 20 min. Meanwhile, the Bio-DotMicrofiltration Apparatus was assembled with a 0.2 μm nitrocellulosemembrane (Bio-Rad Laboratories, Hercules, Calif.) and moistened in 20 mMTris-buffered saline (pH 7.5). Then, the urine supernatant (300 μl)along with increasing concentrations of E. coli-expressed recombinantsurvivin (Li et al., Nature, 1998, 396:580-84) as a standard (0.001-1.0μg/ml) in 300 μl of TBS were filtered onto the membrane. Afterfiltration, the membrane was dried, blocked in 5% Blotto and 0.01%sodium azide in PBS, pH 7.4, for 12 h at 4° C. After washing inPBS-Tween 20 (0.25%), the membrane was incubated with 2 μg/ml of arabbit antibody to survivin (Grossman et al., J. Invest. Dermatol.,1999, 113:1076-81.) for 3 h at 22° C., washed in PBS-Tween, andincubated with a 1:1000 dilution of horseradish peroxidase-conjugateddonkey anti-rabbit IgG (Amersham Biotech, Piscataway N.J.) for 1 h at22° C. After washes in PBS×2 for 10 min, PBS-Tween×2 for 5 min, andPBS×2 for 5 min, binding of the primary antibody was detected byenhanced chemiluminescence (Amersham) and autoradiography. Bands werequantitated by densitometry and a weighted survivin score was calculatedon the basis of the antibody reactivity with increasing concentrationsof recombinant survivin as follows: 0=not detectable; 1=0.001−0.25μg/ml; 2=0.25−1 μg/ml; and 3→1 μg/ml. Each urine specimen was analyzedat least twice on two different occasions and comparable results wereobtained.

Example 2 Western Blotting

[0100] Urine specimens (100 ml) were centrifuged at 1,200×g for 10 minat 22° C., and the cell pellet was washed twice in TBS and solubilizedin 0.5% Triton X-100 in the presence of protease inhibitors for 30 minat 4° C. Samples were separated by SDS gel electrophoresis, transferredto nylon membranes (Millipore, Corp.), and further incubated with 1μg/ml of an antibody to survivin (Grossman et al., J. Invest. Dermatol.,1999, 113:1076-81) followed by horseradish peroxidase (HRP)-conjugatedgoat anti-rabbit IgG and chemiluminescence.

Example 3 RT-PCR

[0101] Fifty milliliters of clean catch urine was obtained from 15patients with new or recurrent urothelial cancer, 2 patients withtreated bladder cancer, 1 patient with prostate cancer, 1 patient withnon-neoplastic urinary tract disease and 1 healthy volunteer. Total RNAwas isolated from urine pellets using the Trizol reagent (LifeTechnologies, Inc., Gaithersburg, Md., U.S.A.). Single strand cDNA wassynthesized by random priming of 1-5 μg total RNA using 1 μl ofSuperscript reverse transcriptase (Gibco BRL, Life Technologies, Inc.,Gaithersburg, Md., U.S.A.) for 1 h at 43° C. After heating at 70° C. for15 min, a first amplification reaction was carried out with survivinprimers 5′-CTGCCTGGCAGCCCTTTCTCAA-3′ (forward; SEQ ID NO: 1) and5′AATAAACCCTGGAAGTGGTGCA-3′ (reverse; SEQ ID NO: 2) with denaturation at94° C. for 15 sec, annealing at 53° C. for 15 sec and extension at 72°C. for 1 min for 20 cycles, followed by incubation at 72° C. for 5 min.A 463-base pair (bp) fragment of the survivin cDNA was subjected to asecond round of amplification with nested survivin primers5′-CCGCATCTCTACATTCAAGAAC-3′ (forward; SEQ ID NO: 3) and5′-CTTGGCTCTTTCTCTGTCC-3′ (reverse; SEQ ID NO: 4), with denaturation at94° C. for 30 sec, annealing at 60° C. for 30 sec, and extension at 72°C. for 45 sec for 30 cycles, followed by incubation at 72° C. for 5 min.The amplified survivin cDNA of 279 bp was separated on a 2.0% agarosegel and visualized by ethidium bromide staining. Control reactions wereamplified using β-actin-specific primers 5′-AGCGGGAAATCGTGCGTG-3′(forward; SEQ ID NO: 5) and 5′-CAGGGTACATGGTGGTGCC-3′ (reverse; SEQ IDNO: 6) with generation of a 309-bp fragment.

Results

[0102] A representative experiment of detection of urine survivin usingthe Bio-Dot test is shown in FIG. 1. Determination of urine survivinwith the Bio-Dot method was carried out in 138 out of the 158 specimenscollected for this study (Table 1). Twenty additional urine samples wereanalyzed for survivin expression by RT-PCR to independently evaluate thespecificity of the Bio-Dot method. Survivin was not detected in urine ofnormal volunteers (0/16), or patients with benign prostate hyperplasia(0/6), interstitial cystitis (0/2), renal calculi (0/3), urinary tractinfection (0/6), or other non-neoplastic urinary tract disease (0/6)(Table 1). Urine survivin was detected in 3 out of 5 patients withcryptogenic hematuria (weighted survivin score, 2), who presented with ahistory of retention and dysuria post trans-urethral prostate resection,and revealed a trabeculated, irregularly thickened bladder, bycystoscopy. One patient with increased PSA levels but without diagnosisof prostate cancer was positive for urine survivin (Table 1). Thispatient also revealed a trabeculated, thickened bladder, by cystoscopy.Survivin was not detected in urine specimens of patients with prostate(0/19), renal (0/8), vaginal (0/1), or cervical (0/1) cancer (Table 1).In contrast, urine survivin was detected in all patients (31/31) withnew onset or recurrent bladder cancer (Table 1). Histopathologic grading(grades I through IV) of the 31 patients in group 4 analyzed for urinesurvivin by Bio-Dot SF included 13 patients with grade II, sevenpatients with grade III, and five patients with grade IV tumors.Carcinoma in situ (CIS) was found in association with the papillary andinvasive carcinomas of 5 patients and in association with high gradeurothelial cancer of the ureter in one patient. TABLE 1 SurvivinDetection in 138 Urine Specimens by the Bio-Dot SF module. Survivin-Survivin- Urine specimens (n) negative positive Group 1 (control healthyvolunteers) 16 16  0 Group 2 (Non-neoplastic urinary tract 29 diseases)Hematuria  5 2 3 UTI  6 6 0 BPH  6 6 0 Rising PSA  1 0 1 Interstitialcystitis  2 2 0 Renal calculi  3 3 0 Other†  6 6 0 Group 3(Genito-urinary cancers except 29 bladder) Prostate 19 19  0 Renal  8 80 Vaginal  1 1 0 Cervical  1 1 0 Group 4 (new or recurrent bladdercancer)  31|7 0 31  Group 5 (treated bladder cancer¶) 33 30   3‡ #tientshad urine cytology positive for bladder cancer.

[0103] Thirty of 33 patients in group 5 analyzed by Bio-Dot SF had nodetectable urine survivin (Table 1). Five of these 30 patients werereceiving BCG and had completed 3-5 treatments, the other 25 were statuspost-treatment with negative cystoscopy. Three patients in group 5 withinitial diagnosis of GII non-invasive bladder cancer tested positive forurine survivin after undergoing negative cystoscopic examination. One ofthe 3 patients had urine cytology positive for bladder cancer. Two ofthe 3 patients were treated with transurethral resection of the bladdertumor and one was treated with fulguration.

[0104] When normalized for a weighted survivin score, patients with CIShad considerably higher survivin score (2.5 . . . 0.5, n=6) thanpatients with grade II bladder cancer (1.3 . . . 0.6, n=13). Thecorrelation between weighted survivin score and histopathology orgrading of the various bladder cancer cases is shown in Tables 2 and 3,respectively. TABLE 2 Correlation Between Weighted Urine Survivin Scoreand Bladder Cancer Histopathology. Histopathology Cases tested Averagesurvivin score ND 3 1.7 ± 1.2 Non-invasive papillary carcinoma 4 1 ± 0No detrusor muscle invasion 12  1.6 ± 0.8 Muscle invasion 6 1.7 ± 0.8CIS 6  2.5 ± 0.5†

[0105] TABLE 3 Correlation Between Weighted Urine Survivin Score andBladder Cancer Grading. Grade Cases tested Average survivin score GradeII 13  1.3 ± 0.6 Grade III 7 1.5 ± 0.8 Grade IV 5 2 ± 1 Grade IV 1 3*#I-IV. ND, not determined. CIS, carcinoma in situ. †Significantlygreater than either Grade II or non-invasive papillary carcinoma (p <0.02).

[0106] By Western blotting, a single survivin band of 16.5 kDa wasdetected in the urine cell pellet from a patient with bladder cancer butnot in that from a healthy volunteer (FIG. 2).

[0107] To independently evaluate the results obtained with the Bio-Dotmethod, 15 additional patients with new or recurrent bladder cancer wereanalyzed for urine survivin by RT-PCR. A 279 bp survivin cDNA wasamplified from urine cell pellets of all the 15 new patients withbladder cancer (15/15) (FIG. 3 and data not shown). In contrast, urinecell pellets from 5 additional individuals, one with urinary tractinfection, two with treated bladder cancer and negative cystoscopy, onewith prostate cancer, and one from a normal volunteer, had no survivincDNA (FIG. 3). In control experiments, a 309 bp β-actin cDNA fragmentwas indistinguishably amplified from urine of controls and patients withbladder cancer (FIG. 3). Histopathologic cases of bladder canceranalyzed by RT-PCR included five patients with grade II tumors, onepatient with grade III, six patients with grade IV and 3 patients withCIS. These experiments suggest that exfoliated cancer cells passivelyrelease survivin in the extracellular milieu, i.e. urine, during tumorprogression.

[0108] In the patient series examined here, the sensitivity of the urinesurvivin test for new or recurrent bladder cancer was 100%, and itsspecificity for other neoplastic and non-neoplastic genito-urinarydiseases was 95% (p<0.02). Because of its high specificity, the urinesurvivin test may be useful to complement cytology and/or otherdiagnostics markers (Ramakumar et al., J. Urol. 1999, 161:388-94;Lokeshwar et al., J. Urol., 2000, 163:348-56) to better monitor bladdercancer patients and identify early recurrences or de novo neoplasms.Other potential advantages of the urine survivin test include itssimplicity, suitability as a point-of-service procedure, and itscost-effectiveness, using one-step detection with a single antibody tosurvivin that has now become commercially available.

Example 4 Detection of Survivin in Blood Serum of Patients

[0109] Blood was collected from bladder and prostate cancer patients.The blood serum was isolated and tested for the presence of survivin bythe dot blot method described in Example 1. Survivin was detected in theblood serum of bladder and prostate cancer patients. Increasingconcentrations of recombinant survivin in μg/ml (left and right column),urine or serum samples were applied to a slot-blot apparatus. Themembrane was incubated with an antibody to survivin followed byHRP-conjugated goat anti rabbit IgG. Bands were visualized bychemiluminescence.

[0110] The detection of survivin in blood serum of cancer patientsindicates that survivin is useful as a marker for detecting othercancers. Testing the serum for the presence of survivin is useful forscreening and for detection of recurrences and relapses of cancer.

[0111] It should be understood that the foregoing discussion andexamples merely present a detailed description of certain preferredembodiments. It therefore should be apparent to those of ordinary skillin the art that various modifications and equivalents can be madewithout departing from the spirit and scope of the invention. Alljournal articles, other references, patents, and patent applicationsthat are identified in this patent application are incorporated byreference in their entirety.

1 6 1 22 DNA Artificial Sequence Description of Artificial SequenceForward PCR primer 1 ctgcctggca gccctttctc aa 22 2 22 DNA ArtificialSequence Description of Artificial Sequence Reverse PCR primer 2aataaaccct ggaagtggtg ca 22 3 22 DNA Artificial Sequence Description ofArtificial Sequence Forward PCR primer 3 ccgcatctct acattcaaga ac 22 419 DNA Artificial Sequence Description of Artificial Sequence ReversePCR primer 4 cttggctctt tctctgtcc 19 5 18 DNA Artificial SequenceDescription of Artificial Sequence Forward PCR primer 5 agcgggaaatcgtgcgtg 18 6 19 DNA Artificial Sequence Description of ArtificialSequence Reverse PCR primer 6 cagggtacat ggtggtgcc 19

1. A method of diagnosing cancer in a patient comprising assaying asample of biological fluid from a patient for the presence or absence ofsurvivin, wherein the presence of survivin in the sample indicates thatthe patient has cancer.
 2. The method of claim 1, wherein the biologicalfluid is urine or blood serum.
 3. The method of claim 1, wherein thecancer is any cancer invading the genitourinary tract.
 4. The method forclaim 3, wherein the genitourinary tract cancer is bladder or prostatecancer.
 5. The method of claim 4, wherein the bladder or prostate canceris graded as a CIS.
 6. The method of claim 4, wherein the bladder orprostate cancer is any grade or any stage.
 7. The method of claim 1,wherein survivin is detected using an agent selected from the groupconsisting of antibodies that bind survivin, survivin binding partners,and nucleic acids that hybridize to a nucleic acid encoding survivin. 8.The method of claim 7, wherein the agent is tagged with a label.
 9. Themethod of claim 8, wherein the label is a radioactive label, afluorescent label, an enzyme, or a chemiluminescent tag.
 10. The methodof claim 1, wherein survivin is detected by an immunoassay.
 11. Themethod of claim 10, wherein the immunoassay is an enzyme linkedimmunosorbent assay or radioimmunoassay.
 12. The method of claim 10,wherein the immunoassay comprises immunoblotting, immunodiffusion,immunoelectrophoresis, or immunoprecipitation.
 13. The method of claim1, wherein survivin is detected by dot blotting.
 14. The method of claim13, wherein dot blotting comprises using a Bio-Dot SF module.
 15. Themethod of claim 1, wherein survivin is detected by nucleic acidhybridization.
 16. The method of claim 15, wherein the nucleic acidhybridization is RT-PCR or Northern blot analysis.
 17. A kit fordiagnosis, prognosis, or monitoring cancer, comprising a container forcollecting biological fluid from a patient and an agent that detects thepresence of survivin in the biological fluid.
 18. The kit of claim 17,wherein the agent is selected from the group consisting of antibodiesthat bind survivin, survivin binding partners, and nucleic acids thathybridize to the nucleic acid encoding survivin.
 19. The kit of claim18, wherein the agent is tagged with a label.
 20. The kit of claim 18,wherein the label is a radioactive label, a fluorescent label, anenzyme, or a chemiluminescent tag.
 21. The kit of claim 17, wherein theagent is packaged in an aqueous medium or in lyophilized form.
 22. Thekit of claim 17, further comprising a means to analyze the presence ofsurvivin.
 23. The kit of claim 17, wherein the cancer is bladder orprostate cancer.
 24. The kit of claim 17, wherein the biological fluidis urine or blood serum.
 25. A method of determining the grade of acancer in a patient comprising quantitating the amount of survivin inthe sample of biological fluid from a patient and comparing the amountof survivin in the sample with the amount of survivin in control samplesto determine the grade of the cancer.
 26. A method of determining thestage of a cancer in a patient comprising quantitating the amount ofsurvivin in the sample of biological fluid from a patient and comparingthe amount of survivin in the sample with the amount of survivin incontrol samples to determine the stage of the cancer.
 27. A method ofmonitoring cancer in a patient comprising quantitating the amount ofsurvivin in the sample of biological fluid from a patient to determinethe grade of the cancer.
 28. The method of claim 1, wherein thebiological fluid is selected from the group consisting of prostaticfluid, seminal fluid, whole blood, serum, urine, breast biopsy fluid,gastrointestinal fluid, and vaginal fluid.
 29. The method of claim 1,wherein the cancer is any cancer that expresses survivin.
 30. The methodof claim 1, wherein the cancer is selected from the group consisting ofneuroblastoma, breast cancer, lung cancer, bladder cancer, colorectalcancer, pancreatic cancer, genitourinary tract cancer, prostate cancer,renal cancer, and bladder cancer.
 31. The method of claim 1, wherein thecancer is new onset cancer or recurrent cancer.